Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype D from non-D genotypes

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http://www.ingentaconnect.com/content/bsc/jvh/2011/00000018/00000004/art00030

Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype

D from non-D genotypes

Authors: Amini-Bavil-Olyaee, S.; Pourkarim, M. R.; Schaefer, S.1; Mahboudi, F.2;

Van Ranst, M.3; Adeli, A.2; Trautwein, C.4; Tacke, F.4

Source: Journal of Viral Hepatitis, Volume 18, Number 4, April 2011 , pp.

300-304(5)

Publisher: Wiley-Blackwell

Abstract:

Summary. 

Hepatitis B virus (HBV) viral load and its genotype play important roles in

clinical outcome, management of disease and response to antiviral therapy. In

many parts of the world such as Europe or the Middle East, distinguishing HBV

genotype D from non-D is most relevant for treatment decisions, because genotype

D-infected patients respond poorly to interferon-based therapeutic regimens.

Here, we developed an in-house real-time PCR to concordantly assess HBV genotype

(D vs non-D) based on melt curve analysis and quantify the viral load. Genotype

distinction was established with control plasmids of all HBV genotypes and

validated with 57 clinical samples from patients infected with six different HBV

genotypes. Our in-house real-time PCR assay could discriminate HBV genotype D

from non-D using single-step melt curve analysis with a 2 °C difference in

the melt curve temperature in all samples tested. Viral load quantification was

calibrated with the WHO HBV international standard, demonstrating an excellent

correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear

range from 3.2 × 102 to 3.2 × 1010 IU/mL. In conclusion, we

developed a rapid, simple and cost-effective method to simultaneously quantify

and distinguish HBV genotypes D from non-D with a single-step PCR run and melt

curve analysis. This assay should be a useful diagnostic alternative to aid

clinical decisions about initiation and choice of antiviral therapy, especially

in geographical regions with a high prevalence of HBV genotype D.

DOI: 10.1111/j.1365-2893.2010.01308.x

Affiliations: 1: Institut für Medizinische Mikrobiologie, Virologie und

Hygiene, Universität Rostock, Schillingallee, Rostock, Germany 2: Biotechnology

Department, Pasteur Institute of Iran, Tehran, Iran 3: Laboratory of Clinical

Virology, Rega Institute for Medical Research, Leuven, Belgium 4: Department of

Medicine III, RWTH-University Hospital Aachen, Aachen, Germany

Publication date: 2011-04-01

[Guest guest]

http://www.ingentaconnect.com/content/bsc/jvh/2011/00000018/00000004/art00030

Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype

D from non-D genotypes

Authors: Amini-Bavil-Olyaee, S.; Pourkarim, M. R.; Schaefer, S.1; Mahboudi, F.2;

Van Ranst, M.3; Adeli, A.2; Trautwein, C.4; Tacke, F.4

Source: Journal of Viral Hepatitis, Volume 18, Number 4, April 2011 , pp.

300-304(5)

Publisher: Wiley-Blackwell

Abstract:

Summary. 

Hepatitis B virus (HBV) viral load and its genotype play important roles in

clinical outcome, management of disease and response to antiviral therapy. In

many parts of the world such as Europe or the Middle East, distinguishing HBV

genotype D from non-D is most relevant for treatment decisions, because genotype

D-infected patients respond poorly to interferon-based therapeutic regimens.

Here, we developed an in-house real-time PCR to concordantly assess HBV genotype

(D vs non-D) based on melt curve analysis and quantify the viral load. Genotype

distinction was established with control plasmids of all HBV genotypes and

validated with 57 clinical samples from patients infected with six different HBV

genotypes. Our in-house real-time PCR assay could discriminate HBV genotype D

from non-D using single-step melt curve analysis with a 2 °C difference in

the melt curve temperature in all samples tested. Viral load quantification was

calibrated with the WHO HBV international standard, demonstrating an excellent

correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear

range from 3.2 × 102 to 3.2 × 1010 IU/mL. In conclusion, we

developed a rapid, simple and cost-effective method to simultaneously quantify

and distinguish HBV genotypes D from non-D with a single-step PCR run and melt

curve analysis. This assay should be a useful diagnostic alternative to aid

clinical decisions about initiation and choice of antiviral therapy, especially

in geographical regions with a high prevalence of HBV genotype D.

DOI: 10.1111/j.1365-2893.2010.01308.x

Affiliations: 1: Institut für Medizinische Mikrobiologie, Virologie und

Hygiene, Universität Rostock, Schillingallee, Rostock, Germany 2: Biotechnology

Department, Pasteur Institute of Iran, Tehran, Iran 3: Laboratory of Clinical

Virology, Rega Institute for Medical Research, Leuven, Belgium 4: Department of

Medicine III, RWTH-University Hospital Aachen, Aachen, Germany

Publication date: 2011-04-01